JP2006000122A - Mutant yeast strain for production of liquor and method for producing liquor using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for improving content of amino acid in production of liquors (especially wine) and a method for improving content of gamma-aminobutyric acid. <P>SOLUTION: Disclosed is a mutant yeast strain for production of liquors decreasing the functions of gap1, apf1(shr3), can1, put4 and uga4, wherein the mutant yeast strain is obtained by crossbreeding a mutant yeast strain decreasing the functions of gap1, apf1(shr3) and can1 with a mutant yeast strain decreasing the functions of gap1, put4 and uga4 to have resistance against L-azetidine-2-carboxylate and L-canavanine, and wherein the mutant yeast strain can not grow in a minimal medium in which proline, arginine, glutamic acid, citruline or gamma-aminobutyric acid is added as a single nitrogen resource. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a yeast used for brewing alcoholic beverages, and to a method for producing alcoholic beverages using yeast.

  In general, amino acids are one of the taste components of alcoholic beverages such as wine, and are known to be important factors affecting quality. Therefore, it is important in the production of alcoholic beverages to control the amino acid content in the produced alcoholic beverages.

  However, for example, in the production of wine, most of the amino acids in the wine produced are from the grape juice, but many of the amino acids in the grape juice are consumed during fermentation as a nutrient source for yeast. It is extremely difficult to control the amino acid content in wine.

  The wine produced from Koshu grape, which is a typical grape varieties produced in Japan, has a relatively flat taste and is thought to be caused by the low content of nitrogen compounds including amino acids. ing. Therefore, Koshu wines are mainly sweet wines that leave sugar to supplement the flat taste. When producing dry wines, the Shu-Lee method, that is, by contacting the wine with starch, A method of giving a sense of body to wine by eluting components such as amino acids from self-digestion (K. Ariizumi et al .: Am. J. Enol. Vitic., Vol.45, pp.312, 1994) . However, the Sur-Lee method has a problem that the contact period between wine and starch is long, and further, the increase in amino acids in wine due to yeast autolysis is not always large. In recent years, diversification of liquor quality has been demanded, and in order to meet this demand, development of a new liquor production method capable of controlling the amino acid content in the produced liquor is desired.

By the way, in order for yeast to use an amino acid present outside the cell as a nutrient source, it is essential to incorporate the amino acid into the microbial cells. There are many amino acid permeases with different substrate specificities, such as general amino acid permease encoded by the GAP1 gene and arginine permease encoded by the CAN1 gene. (B. Andre: Yeast, vol. 11, pp. 1575, 1995, B. Nelissen et al .: FEBS Letters, vol. 377, pp. 232, 1995, M. Grenson: In De Pont, JJHHM (ed.), Molecular Aspects of Transport Proteins, Chapter 7, “Amino acid transporters in yeast; structure, function and regulation”, Elsevier, Amsterdam, pp. 219, 1992, etc.).

So far, there have been many reports on methods for obtaining mutant strains in which mutations have been made in genes involved in the amino acid permease of yeast and the effects of the mutations on amino acid incorporation. For example, gap1 mutant strains can be obtained from strains resistant to D-amino acids, and it is known that incorporation of many amino acids including citrulline is suppressed (J. Rytka: J. Bacteriol. , vol.121, pp.562, 1975). The can1 mutant strain can be obtained from a strain resistant to L-canavanine, an analog of arginine, and is known to have suppressed arginine uptake (M. Grenson et al .: Biochem. Biophys Acta, vol. 127, pp. 325, 1966, M. Grenson, Molecular Aspects of Transport Proteins, Chapter 7, “Aminoacid transporters in yeast: structure, function and regulation, Elsevier Science Publishers, 1992, etc. ) and apf1 mutants Can be obtained from strains resistant to L-azetidine-2-carboxylate or 3,4-dihydro-DL-proline, which are analogs of proline. activity is known to be inhibited, further, this in the yeast with a double mutation gap1 and APF 1, which can not grow a number of amino acids, including glutamic acid as the sole nitrogen source (M. Grenson and C. Hennaut: J. Bacteriol., Vol.105, pp.477, 1971, PF Lasko and MC Brandriss: J. Bacteriol., Vol.148, pp.241, 1981) .

The SHR3 gene encodes a protein essential for processing and transport of amino acid permease from the endoplasmic reticulum, and shr3 and apf1 mutations have been reported to be non-complementary alleles (PO Ljungdahlet al .: Cell, vol. 71, pp. 463, 1992, J. Horak and A. Kotyk: Biochemistry and Molecular Biology International, vol. 29, pp. 907, 1993).

  On the other hand, γ-aminobutyric acid is one of the non-protein constituent amino acids widely present in the living world, and has a brain metabolism promoting action as a major inhibitory transmitter of nerves and a blood pressure lowering action. Various foods and food materials are being developed, including tea leaves (gabalon tea) in which γ-aminobutyric acid is accumulated by anaerobic treatment using carbon dioxide and nitrogen gas (Tushida et al., Agricultural, 61, 817 (1987), Omori et al., Agricultural, 61, 1449 (1987), JP-A-10-215812, JP-A-9-238650, JP-A-7-213252, etc.).

By the way, although γ-aminobutyric acid is also contained in grape juice, it is consumed as a nutrient source for yeast during fermentation as well as other amino acids, so its content in wine is very small. It was difficult to leave many. Incidentally, incorporation of γ- aminobutyric acid by yeast by general amino acid permease, PUT4 proline permease and 3 kinds of amino acid transmission enzyme γ-aminobutyric acid permease encoded by the UGA4 gene encoded by the gene encoded by the GAP1 gene (M. Glenson et al .: Biochem. (Life Sci. Adv.), Vol. 6, pp. 35, 1987).

JP-A-10-215812 JP-A-9-238650 JP-A-7-213252 B. Andre: Yeast, vol.11, pp.1575, 1995 B. Nelissen et al .: FEBS Letters, vol.377, pp.232, 1995 M. Grenson: In De Pont, J. J. H. H. M. (ed.), Molecular Aspects of Transport Proteins, Chapter 7, "Amino acid transporters in yeast; structure, function and regulation", Elsevier, Amsterdam, pp. 219, 1992 M. Grenson et al .: Biochem. Biophys. Acta, vol. 127, pp. 325, 1966 M. Grenson and C. Hennaut: J. Bacteriol., Vol.105, pp.477, 1971 P. F. Lasko and M. C. Brandriss: J. Bacteriol., Vol.148, pp.241,1981 P. O. Ljungdahl et al .: Cell, vol.71, pp.463, 1992 J. Horak and A. Kotyk: Biochemistry and Molecular Biology International, vol. 29, pp. 907, 1993 Tsushida et al., Agricultural Chemistry, 61, 817 (1987) Omori et al., Agricultural Chemistry, 61, 1449 (1987) M. Glenson et al .: Biochem. (Life Sci. Adv.), Vol.6, pp.35, 1987

  The present invention has been made in view of the above-mentioned problems of the prior art, and provides a method for improving the content of amino acids and the content of γ-aminobutyric acid in the production of alcoholic beverages (especially wine). It aims to provide a way to do.

As a result of intensive studies to achieve the above object, the present inventors have found that genes involved in amino acid incorporation, namely, mutations in gap1 , apf1 ( shr3) and can1 , mutations in gap1 , put4 and uga4 , gap1 , apf1 ( The present inventors have found that the amino acid content is improved in alcoholic beverages brewed using a yeast mutant for producing alcoholic beverages having shr3) , can1 , put4 and uga4 mutations, and the present invention has been completed.

That is, the yeast mutant for producing alcoholic beverages of the present invention belongs to the genus Saccharomyces, and is a yeast mutant for producing alcoholic beverages having mutations of gap1 , apf1 ( shr3) and can1 as mutations of genes involved in amino acid uptake, for example, Saccharomyces cerevisiae MA3911 (FERM P-17839) is preferred.

In addition, the yeast mutant strain for producing alcoholic beverages of the present invention belongs to the genus Saccharomyces, and is a yeast mutant strain for producing alcoholic beverages having mutations of gap1 , put4 and uga4 as mutations of genes involved in amino acid incorporation, for example, Saccharomyces S. cerevisiae MA3719 (FERM P-17838) is preferred.

Furthermore, the yeast mutant strain for producing alcoholic beverages of the present invention belongs to the genus Saccharomyces and has a mutation of gap1 , apf1 ( shr3) , can1 , put4 and uga4 as a mutation of a gene involved in amino acid incorporation. For example, Saccharomyces cerevisiae YMK-7 (FERM P-17837) is preferable.

  The method for producing alcoholic beverages of the present invention is a method for producing alcoholic beverages characterized in that the above-described yeast mutant of the present invention is used in a method for producing alcoholic beverages using yeast.

  Here, in the method for producing an alcoholic beverage of the present invention, the alcoholic beverage is preferably wine or a malt alcoholic beverage.

The method for producing an alcoholic beverage of the present invention also includes adding ammonium phosphate or ammonium sulfate to promote the promotion of fermentation, and the mutations in gap1 , apf1 ( shr3) and can1 or gap1 , apf1 ( shr3) , can1 , put4 and uga4 The method for producing alcoholic beverages is characterized by inoculating and fermenting a bacterial strain having sucrose. In particular, the method for producing alcoholic beverages has an improved amino acid content.

In addition, the wine production method of the present invention uses fermented grapes as an ingredient, inoculating and fermenting strains having mutations of gap1 , put4 and uga4 or gap1 , apf1 ( shr3) , can1 , put4 and uga4. In particular, it is a wine production method in which the content of γ-aminobutyric acid in the wine to be produced is improved.

  According to the present invention, it becomes possible to produce alcoholic beverages (especially wines) in which the amino acid content is stably improved by suppressing the uptake of amino acids by yeast, and the content of γ-aminobutyric acid. It becomes possible to produce alcoholic beverages (especially wines) with improved.

The present invention belongs to the genus Saccharomyces, and yeast mutants having a plurality of mutations in genes involved in amino acid incorporation, particularly yeast mutants for liquor production to which mutations of gap1 , apf1 ( shr3) and can1 , have been added , gap1 , The present invention relates to a yeast mutant for producing alcoholic beverages to which mutations of put4 and uga4 are imparted, and a yeast mutant for producing alcoholic beverages to which mutations of gap1 , apf1 ( shr3) , can1 , put4 and uga4 are imparted.

  The present invention also relates to a method for producing an alcoholic beverage with improved amino acid content, particularly a wine, characterized by inoculating a yeast mutant strain having the above mutation as a liquor and performing fermentation.

The yeast used in the present invention is a yeast belonging to the genus Saccharomyces, for example, Saccharomyces cerevisiae classified according to The Yeasts A Taxonomic Study, 4th ed. (CP Kurtzman and JW Fell (ed.), Elsewier (1998)). (Saccharomyces cerevisiae), Saccharomyces Baianasu (Saccharomyces bayanus), include wine yeast and / or brewer's yeast belonging to Saccharomyces pastorianus (Saccharomyces pastorianus) or the like. Examples of yeast belonging to Saccharomyces cerevisiae include wine yeasts such as Saccharomyces cerevisiae OC-2 strain, W3 strain, and Prize de Mousse strain.

  As a method for mutating yeast, there are a physical method such as ultraviolet irradiation or a chemical method of suspending in a solution of a mutating agent such as ethyl methanesulfonate, and a known method can be appropriately used.

  Hybridization between yeasts can be easily performed by spore-spore, spore-cell, or cell-cell junction as appropriate in consideration of the mating type and ploidy. By inducing ascospore formation with sodium acetate 0.82%, glucose 0.1%, yeast extract 0.25%, potassium chloride 0.18%, agar 2%) (% = w / v%), separating spores and forming colonies Single spore strains can be isolated. Moreover, the target yeast mutant can be efficiently obtained by appropriately combining the mutation treatment and the method of hybridization between the mutants.

Next, a method for obtaining a yeast mutant will be described. The gap1 mutant can be obtained from strains that are resistant to D-amino acids, such as D-histidine, after inducing mutation in yeast. That is, from a mutant strain showing resistance to D-histidine, a minimal medium supplemented with citrulline as a single nitrogen source (glucose 0.5%, Difco Bacto yeast nitrogen base (without amino acids and ammonium sulfate) 0.17%, agar 2 %) (% = W / v%) and can be obtained by selecting a strain having no complementarity in a complementation test using a known gap1 mutant strain. The can1 mutant can be obtained from a strain exhibiting resistance to L-canavanine, which is an analog of arginine, and can be obtained by selecting a strain having no complementarity in a complementation test using a known can1 mutant. The apf1 mutant can be obtained from strains resistant to proline analogs L-azetidine-2-carboxylate or 3,4-dihydro-DL-proline and resistant to β-2-chenyl-DL-alanine It can be obtained by selecting a strain that cannot grow on a medium supplemented with proline as a single nitrogen source and has no complementarity in a complementation test using a known apf1 mutant strain.

In addition, in order to obtain double mutant strains from the mutant strain thus obtained, a method of remutating the mutant strain obtained above or a hybrid strain between mutant strains, A method of selecting from the single spore strains can be used. The above-described method can be used when selecting a target mutant strain from the subsequent mutation treatment. For example, the mutant strain having gap1 and apf1 mutations may be subjected to the mutation treatment again using the strain already having the gap1 mutation, and the above-described method for obtaining the apf1 mutant strain may be performed.

A strain having three or more mutations can be obtained in the same manner as a strain having double mutations, and strains having gap1 , apf1 ( shr3) and can1 mutations have, for example, gap1 and apf1 ( shr3) mutations. Cross between strains and strains having gap1 and can1 mutations, and resistant to L-azetidine-2-carboxylate and L-canavanine from single spore strains isolated from hybrid strains by the method described above And a minimal medium supplemented with proline or arginine as a single nitrogen source (glucose 0.5%, Difco Bacto yeast nitrogen base (without amino acids and ammonium sulfate) 0.17%, agar 2%) (% = By selecting a strain that is not capable of growing at w / v%) and does not have complementarity in a complementation test with a known strain of gap1 , apf1 ( shr3) or can1, respectively. Can be obtained. In addition, strains having mutations of gap1 , put4, and uga4 are, for example, induced mutations of gap1 and put4 among strains having resistance to L-azetidine-2-carboxylate after inducing mutations in strains having gap1 mutation. After selecting a strain having the same , and after performing a mutation treatment again, it cannot grow on a minimal medium supplemented with γ-aminobutyric acid as a single nitrogen source, and has known gap1 , put4 and uga4 mutations In a complementation test with a strain, it can be obtained by selecting a strain having no complementarity. Further , strains having mutations of gap1 , apf1 ( shr3) , can1 , put4 and uga4 are, for example, strains having the mutations of gap1 , apf1 ( shr3) and can1 obtained by the method described above and mutations of gap1 , put4 and uga4 . Can be obtained by crossing between strains having

Examples of the strains used in the acquisition of the mutant strains, for example, MA1007 strain having gap1 mutation obtained from Saccharomyces cerevisiae OC-2 strain of the, MA2504 has a mutation gap1 and apf1 (shr3) (FERMP-17407 ) Strain, MA2601 (FERMP-17408) strain having mutations in gap1 and can1 .

  The yeast mutant of the present invention can be suitably used for the production of alcoholic beverages such as wine and malt alcoholic beverages (for example, beer and foamed liquor), and particularly preferably for the production of wine.

  In order to produce wine in the present invention, the obtained yeast mutant strain is used as a liquor mother, and if necessary, ammonium phosphate or ammonium sulfate is added to grape juice at 200 to 2000 mg / l, preferably 500 to 1000 mg / l, Wines can be brewed by a method similar to a commonly used wine production method, for example, the method described in the sake brewing method (Yamanashi Prefectural Industrial Technology Center). When wine is produced using grapes that have been anaerobically treated, the grapes should be stored under carbon dioxide or nitrogen gas for 6 hours to 240 hours (10 days), preferably 24 hours to 72 hours at room temperature. Good. In the case of producing red wine, the Maseracion Carbonic method, in which grapes that are not subjected to infarction and crushing, are placed in a sealed tank and replaced with carbon dioxide in the tank, is also a method that has the same effect as anaerobic treatment. Applicable.

  In order to produce alcoholic beverages other than wine in the present invention, the obtained yeast mutant strain is used, and a method similar to the usual method for producing alcoholic beverages, for example, “Technology Brewing and Malting”, by Kunze, VLB, 1996 Year: “Die Bierbrauerei, Band I, II”, (7th edition), Narziss, Enke, 1992; “Brew brewing” by Matsuyama Shigesuke, Toyo Keizai Shinposha, 1969. .

  In the production of alcoholic beverages using the yeast mutant of the present invention, since the incorporation of amino acids is suppressed, it is possible to produce wines with a stable improvement in amino acid content.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to a following example.

Examples 1-3 and Comparative Examples 1-5
(Acquisition of yeast mutant)
MA-12504 (FERM P-17407) strain (comparative example 2) having gap1 and apf1 ( shr3) mutations obtained from the wine yeast Saccharomyces cerevisiae OC-2 strain (Comparative Example 1) and gap1 and can1 mutations Each of MA-2601 (FERM P-17408) strain (Comparative Example 3) was induced to form ascospores using a medium of McClary et al., And phosphomoles containing 250 ppm of Zymolyace (trade name: Zymolyce 20T, manufactured by Seikagaku Corporation) After the ascending wall was dissolved in an acid buffer (pH 7.5), sexual crossing was performed between spores and spores using a micromanipulator. The resulting hybrid strain again formed ascospores in the medium of McClary et al., Dissolved the ascomal wall in a phosphate buffer solution (pH 7.5) containing 250 ppm zymolyce, and then used a micromanipulator Single spores were obtained by separating offspring. Among the obtained single spore strains, it is the least resistant to L-azetidine-2-carboxylate and L-canavanine and added with amino acids of proline, arginine, glutamic acid and citrulline as a single nitrogen source. The MA3911 strain (Example 1) that cannot grow in the medium was selected as a strain having the mutations of gap1 , apf1 ( shr3), and can1 .

Further, strains with known gap1 mutation, for example, 2512c-2A (a gap1) in minimal medium containing citrulline single nitrogen source in crosses with the growth is not observed, strains with known apf1 mutations, e.g. Resistant to L-azetidine-2-carboxylate in a cross with 30967c (a apf1 ura3 ), in a cross with a strain having a known can1 mutation, for example, AH-22 (a leu2-3,112 his4can1 ) It was confirmed to have the target mutation by showing resistance to L-canavanine, that is, lack of complementation ability. This strain was deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology on April 27, 2000, and the deposit number is FERM P-17839.

In addition, the MA1007 strain (Comparative Example 4) having the gap1 mutation obtained from the wine yeast Saccharomyces cerevisiae OC-2 strain was inoculated into the medium of McClary et al. To induce the formation of ascospores, and this was treated with zymolyce. The ascending wall was dissolved and then washed with sterilized water, and phosphate buffer (pH 8.0) was added to obtain 4.6 ml of a spore suspension. To this, 0.25 ml of 40% glucose solution and 0.15 ml of ethyl methanesulfonate were added and subjected to mutation treatment at 30 ° C. for 80 minutes, and then the mutation treatment was stopped by adding an equal amount of 10% sodium thiosulfate solution. It was washed with sterilized water and suspended in sterilized water to obtain a mutation-treated spore suspension. The mutated spore suspension was spread on a minimal medium containing 0.5 mM L-azetidine-2-carboxylate and 0.1% urea and cultured for 3-7 days. Thereafter, the MA2155 strain (Comparative Example 5) that did not grow on a minimal medium containing proline as a single nitrogen source was selected from the grown colonies as a strain having gap1 and put4 mutations.

Furthermore, the spore suspension obtained by subjecting this selected strain to the same mutation treatment again was diluted with sterilized water as appropriate and applied to a YPD (glucose 2%, yeast extract 2%, polypeptone 1%) medium. After culturing for 3 days, MA3719 strain (Example 2) in which growth was not observed was isolated as a strain having mutations of gap1 , put4 and uga4 by replicating to a minimal medium using γ-aminobutyric acid as a single nitrogen source. . This strain is crossed with a known gap1 , put4 and uga4 mutation, for example 22574d (a gap1 put4 uga4 ura3 ), and growth is not observed in a minimal medium using γ-aminobutyric acid as a single nitrogen source, That is, it was confirmed that the target mutation was present due to lack of complementarity. This strain was deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology on April 27, 2000, and its deposit number is FERM P-17838.

Furthermore, a single spore strain derived from the hybrid strain was obtained by the above-mentioned method from the MA3911 strain having the mutations of gap1 , apf1 ( shr3) and can1 obtained as described above and the MA3719 strain having the mutations of gap1 , put4 and uga4. Among the spore strains, in a minimal medium that is resistant to L-azetidine-2-carboxylate and L-canavanine and supplemented with proline, arginine, glutamic acid, citrulline, and γ-aminobutyric acid as a single nitrogen source The YMK-7 strain (Example 3) that cannot grow is selected as a strain having gap1 , apf1 ( shr3) , can1 , put4, and uga4 mutations, and has the target mutation by a complementary test similar to the above. confirmed. This strain was deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology on April 27, 2000, and its deposit number is FERM P-17837.

  Here, the procedure shown above is shown as a flowchart in FIG. In addition, Table 1 shows the results regarding the presence or absence of growth when the above mutant strains were cultured in a minimal medium supplemented with various amino acids as a single nitrogen source.

gap1 and apf1 (shr3) from mutations in hybrids with MA2601 strain having mutations MA2504 strain and gap1 and can1 with the gap1, apf1 (shr3) and MA3911 strain proline having a mutation of can1, glutamic acid, alanine, No growth was observed in a minimal medium containing arginine and citrulline as a single nitrogen source. On the other hand, the MA2155 strain with the gap1 and put4 mutations obtained from the mutation treatment of the MA1007 strain with the gap1 mutation did not grow in a minimal medium with proline and citrulline as the single nitrogen source, In the MA3719 strain having the mutations of gap1 , put4 and uga4 obtained from the mutation treatment, no growth was observed in a minimal medium containing proline, citrulline and γ-aminobutyric acid as a single nitrogen source. Further, gap1, apf1 (shr3) and MA3911 strain and gap1 having a mutation of can1, PUT4 and gap1, from mutations in hybrids with MA3719 strain having the uga4 apf1 (shr3), the mutation of can1, PUT4 and Uga4 In the YMK-7 strain possessed, all of proline, glutamic acid, alanine, citrulline, arginine, and γ-aminobutyric acid did not grow in a medium using these as a single nitrogen source.

Examples 4-5 and Comparative Examples 6-7
MA3911 strain (Example 4) having the mutations of gap1 , apf1 ( shr3) and can1 obtained above, YMK-7 strain (Example 5) having the mutations of gap1 , apf1 ( shr3) , can1 , put4 and uga4 and Using Saccharomyces cerevisiae OC-2 strain (Comparative Example 6), wine was produced by a conventional method using Koshu grape juice having a sugar content adjusted to 22 degrees with granulated sugar. In addition, the above-mentioned mutant strains have suppressed the uptake of various amino acids, and since the fermentation is slow due to lack of nitrogen in the raw juice, ammonium phosphate is added to the juice at a concentration of 1000 mg / l in advance. Went. Table 2 shows the results of general component analysis of the wines produced.

  The fermentation days of MA3911 strain and YMK-7 strain were almost the same as the control group (OC-2 strain-free group (Comparative Example 7)), and smooth fermentation was obtained. In addition, no significant difference was found in the analysis values of alcohol concentration, extract content, pH, total acid, and organic acid composition of wine from each test section. Table 3 shows the amino acid analysis results.

  Wines brewed with the OC-2 strain were mostly proline, but wines brewed with the MA3911 strain also contained many amino acids such as aspartic acid, threonine, serine, asparagine, glutamic acid, glutamine, alanine, and arginine. The wine brewed with the YMK-7 strain contained a large amount of γ-aminobutyric acid in addition to these amino acids. The total amount of amino acids contained in wines brewed by the MA3911 strain and YMK-7 strain was about twice that of the wine brewed by the OC-2 strain.

Example 6 and Comparative Example 8
The contents of γ-aminobutyric acid and glutamic acid in grape fruits when Muscat Berry A grapes were stored at room temperature under carbon dioxide or air were examined. The results are shown in FIGS. 2 (a) and (b). Under air, there was no significant change in the content of γ-aminobutyric acid and glutamic acid. However, glutamic acid significantly decreases under carbon dioxide, whereas γ-aminobutyric acid increases, and the content of γ-aminobutyric acid when stored for 48 hours is about twice that when stored under air. there were. It was confirmed that γ-aminobutyric acid was also accumulated in grape fruits by anaerobic treatment in the same manner as tea leaves.

Then 48 hours anaerobic treatment under carbon dioxide γ- aminobutyric acid juice was collected from the accumulated allowed the Muscat Berry A grape, by adjusting the sugar content to 21 degrees with granulated sugar, this gap1, MA3719 strain (Example 6) having a mutation of put4 and uga4 and Saccharomyces cerevisiae OC-2 strain (Comparative Example 8) were inoculated as a liquor mother to produce wine. Table 4 shows the general component analysis results of the wines produced.

  Fermentation with the MA3719 strain was smooth, and the fermentation days were almost the same as the OC-2 strain. In addition, no significant difference was found in the analytical values of wine alcohol concentration, extract content, pH, total acid, and organic acid composition. The results of amino acid analysis are shown in Table 5.

  In the wine produced using the OC-2 strain, most of the amino acids contained were proline and the content of γ-aminobutyric acid was small, but the wine produced using the MA3719 strain was added to proline. A large amount of γ-aminobutyric acid was also contained, and the total amino acid content was 2.3 times that of wine produced using the OC-2 strain.

It is a flowchart which shows suitable embodiment of the process of obtaining the yeast mutant concerning this invention. (a) is a graph showing the relationship between the treatment time and the content of γ-aminobutyric acid when Muscat Berry A grapes are stored under carbon dioxide or air. (b) is a graph showing the relationship between the treatment time and glutamic acid content when muscat berry A grapes are stored under carbon dioxide or air.

Claims (6)

L-azetidine-2-carboxyl obtained by crossing a yeast strain having a mutation that reduces the function of gap1 , apf1 ( shr3) and can1 with a yeast strain having a mutation that reduces the function of gap1 , put4 and uga4 Resistant to rate and L-canavanine and unable to grow in minimal medium supplemented with proline, arginine, glutamic acid, citrulline, γ-aminobutyric acid as a single nitrogen source, gap1 , apf1 ( shr3) , can1 , A yeast mutant for alcoholic beverages belonging to the genus Saccharomyces having a mutation that reduces the functions of put4 and uga4 .   The yeast mutant for alcoholic beverage production according to claim 1, wherein the yeast mutant is Saccharomyces cerevisiae YMK-7 (FERM P-17837).   A method for producing alcoholic beverages using the yeast mutant according to claim 1 or 2 in a method for producing alcoholic beverages using yeast.   The method for producing an alcoholic beverage according to claim 3, wherein the alcoholic beverage is a wine or a malt alcoholic beverage.   The method for producing an alcoholic beverage according to claim 3 or 4, wherein in the method for producing an alcoholic beverage, fermentation is performed by adding ammonium phosphate or ammonium sulfate. A method for producing wine, characterized in that an anaerobic-treated grape is used as a raw material, and the yeast mutant according to claim 1 or 2 is inoculated as a liquor and fermented.

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